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Neutron structure of human carbonic anhydrase II: implications for proton transfer.

ABSTRACT: Human carbonic anhydrase II (HCA II) catalyzes the reversible hydration of carbon dioxide to form bicarbonate and a proton. Despite many high-resolution X-ray crystal structures, mutagenesis, and kinetic data, the structural details of the active site, especially the proton transfer pathway, are unclear. A large HCA II crystal was prepared at pH 9.0 and subjected to vapor H-D exchange to replace labile hydrogens with deuteriums. Neutron diffraction studies were conducted at the Protein Crystallography Station at Los Alamos National Laboratory. The structure to 2.0 A resolution reveals several interesting active site features: (1) the Zn-bound solvent appearing to be predominantly a D(2)O molecule, (2) the orientation and hydrogen bonding pattern of solvent molecules in the active site cavity, (3) the side chain of His64 being unprotonated (neutral) and predominantly in an inward conformation pointing toward the zinc, and (4) the phenolic side chain of Tyr7 appearing to be unprotonated. The implications of these details are discussed, and a proposed mechanism for proton transfer is presented.

SUBMITTER: Fisher SZ

PROVIDER: S-EPMC2893723 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Neutron structure of human carbonic anhydrase II: implications for proton transfer.

Fisher S Zoë SZ Kovalevsky Andrey Y AY Domsic John F JF Mustyakimov Marat M McKenna Robert R Silverman David N DN Langan Paul A PA

Biochemistry 20100101 3

Human carbonic anhydrase II (HCA II) catalyzes the reversible hydration of carbon dioxide to form bicarbonate and a proton. Despite many high-resolution X-ray crystal structures, mutagenesis, and kinetic data, the structural details of the active site, especially the proton transfer pathway, are unclear. A large HCA II crystal was prepared at pH 9.0 and subjected to vapor H-D exchange to replace labile hydrogens with deuteriums. Neutron diffraction studies were conducted at the Protein Crystallo ...[more]

PMID: 20025241

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Project description:Catalysis by the zinc metalloenzyme human carbonic anhydrase II (HCA II) is limited in maximal velocity by proton transfer between His64 and the zinc-bound solvent molecule. Asn62 extends into the active site cavity of HCA II adjacent to His64 and has been shown to be one of several hydrophilic residues participating in a hydrogen-bonded solvent network within the active site. We compared several site-specific mutants of HCA II with replacements at position 62 (Ala, Val, Leu, Thr, and Asp). The efficiency of catalysis in the hydration of CO 2 for the resulting mutants has been characterized by (18)O exchange, and the structures of the mutants have been determined by X-ray crystallography to 1.5-1.7 A resolution. Each of these mutants maintained the ordered water structure observed by X-ray crystallography in the active site cavity of wild-type HCA II; hence, this water structure was not a variable in comparing with wild type the activities of mutants at residue 62. Crystal structures of wild-type and N62T HCA II showed both an inward and outward orientation of the side chain of His64; however, other mutants in this study showed predominantly inward (N62A, N62V, N62L) or predominantly outward (N62D) orientations of His64. A significant role of Asn62 in HCA II is to permit two conformations of the side chain of His64, the inward and outward, that contributes to maximal efficiency of proton transfer between the active site and solution. The site-specific mutant N62D had a mainly outward orientation of His64, yet the difference in p K a between the proton donor His64 and zinc-bound hydroxide was near zero, as in wild-type HCA II. The rate of proton transfer in catalysis by N62D HCA II was 5% that of wild type, showing that His64 mainly in the outward orientation is associated with inefficient proton transfer compared with His64 in wild type which shows both inward and outward orientations. These results emphasize the roles of the residues of the hydrophilic side of the active site cavity in maintaining efficient catalysis by carbonic anhydrase.

Dataset Information

Neutron structure of human carbonic anhydrase II: implications for proton transfer.

Publications

Neutron structure of human carbonic anhydrase II: implications for proton transfer.

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